Project Summary: Synaptic dysfunction has been implicated in many neurological diseases including epilepsy, Alzheimer?s, Parkinson?s, autism spectrum disorder (ASD), schizophrenia, depression, ADHD and Huntington?s. Despite the prevalence and severity of these disorders, the development of new therapeutics has lagged. This is due, in part, to challenges in replicating relevant biology in robust, scalable in vitro assays. Current methods of measuring synaptic function, which stimulate presynaptic cells and record from postsynaptic cells, lack sufficient throughput for drug screening. The Optopatch platform recently developed at Q-State Biosciences, comprised of engineered optogenetic proteins, custom microscopes, and software, makes it possible to simultaneously stimulate (blue light) and record (red light) electrical activity from ~100 neurons with 1 millisecond temporal resolution, single-cell spatial resolution and high signal-to-noise ratio. Additionally, patterned blue light can be used to probe synaptic connections by stimulating individual neurons while recording postsynaptic potentials (PSPs) in all remaining cells. In Phase I, we developed synaptic assays in primary rodent neurons for: 1. Presynaptic calcium ? The red calcium sensing protein jRGECO1a is targeted to presynaptic boutons by fusion with synaptophysin. Neural activity is stimulated with blue light via a channelrhodopsin, CheRiff. 2. Postsynaptic calcium ? jRGECO1a is targeted to postsynaptic spines by fusion with PSD95. Distinct subsets of neurons express either actuator or reporter. Action potentials triggered in presynaptic cells generate calcium signals in postsynaptic cells. 3. Postsynaptic voltage ? CheRiff and the red voltage sensing protein QuasAr are expressed in distinct subsets of neurons. Presynaptic cell stimulation leads to PSPs recorded in QuasAr-expressing postsynaptic cells. Pharmacological probes isolate excitatory signaling through either AMPA or NMDA channels or inhibitory signaling through GABAA channels. Inhibitory neurons can be labeled with a fluorescent tag expressed under control of the Dlx1/2 promoter, to resolve different synapse classes: excitatory (E) ? inhibitory (I), E ?E, I ?E, and I ? I. In the follow-on Phase II project, we propose to: (1) transition the assays to human induced pluripotent stem cell derived neurons, testing multiple strategies to increase the synaptic maturation of the cells, (2) expand assays in rodent cells to include plasticity, particularly long-term potentiation (LTP) and spike timing dependent plasticity, and (3) apply these assays in disease models of ASD using knockout of three synaptic proteins, SHANK3, SYNGAP1, and GRIN2B, whose loss causes severe ASD in all cases. The most robust phenotype will be used to (4) screen a library of approved drugs to demonstrate assay throughput and sensitivity and identify candidates for potential repurposing. The establishment of ASD-associated cellular phenotypes for HTS would provide a foundation for drug discovery for these serious and poorly treated diseases.